Fusion proteins, uses thereof and processes for producing same

ABSTRACT

This invention provides fusion proteins comprising consecutive amino acids which beginning at the amino terminus of the protein correspond to consecutive amino acids present in (i) a cytomegalovirus human MHC-restricted peptide, (ii) a first peptide linker, (iii) a human β-2 microglobulin, (iv) a second peptide linker, (v) a HLA-A2 chain of a human MHC class I molecule, (vi) a third peptide linker, (vii) a variable region from a heavy chain of a scFv fragment of an antibody, and (viii) a variable region from a light chain of such scFv fragment, wherein the consecutive amino acids which correspond to (vii) and (viii) are bound together directly by a peptide bond or by consecutive amino acids which correspond to a fourth peptide linker, wherein the antibody from which the scFv fragment is derived specifically binds to mesothelin.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/526,667 filed on Oct. 29, 2014, which is a continuation of U.S.patent application Ser. No. 12/972,560 filed on Dec. 20, 2010, which isa continuation of U.S. patent application Ser. No. 11/804,541 filed onMay 17, 2007, now U.S. Pat. No. 7,977,457, which claims the benefit ofpriority of U.S. Provisional Patent Application No. 60/801,798 filed onMay 19, 2006. The contents of the above Applications are allincorporated herein by reference.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 69510SequenceListing.txt, created on Apr. 6,2017, comprising 21,291 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

Throughout this application, certain publications are referenced. Fullcitations for these publications may be found immediately preceding theclaims. The disclosures of these publications are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention relates.

According to current immune surveillance theory, the immune systemcontinuously locates and destroys transformed cells. However, some cellsescape from an apparently effective immune response and consequentlybecome tumors (1-4). Tumor evasion from immune response is a wellestablished phenomenon demonstrated in numerous studies and is caused bya wide variety of suggested mechanisms (1-4). Among these mechanismsare: the production of suppressive cytokines, the loss of immunodominantpeptides, the resistance to killing mechanisms (apoptosis), and the lossof MHC class I (1-4). One of the evasion mechanisms shown to be stronglycorrelated with tumor progression is the loss or down regulation of MHCclass I molecules. This evasion mechanism is abundant in many tumors andcan result from a number of different mutations. Several studiesrevealed weak spots in the MHC class I loading and presentation routeincluding loss of beta-2-microglobulin, TAP1/TAP2 mutations, LMPmutations, loss of heterozygocity in the MHC genes, and down regulationof specific MHC alleles.

Current cancer immunotherapy strategies typically employ the two arms ofthe immune system: the humoral and the cellular systems. In the first,systemic injection of high affinity monoclonal antibodies (mAbs)directed against cell surface tumor associated antigens has demonstratedstatistically significant anti-tumor activity in clinical trials (5,6).Furthermore, anti-tumor mAbs that carry effectors such as cytokines ortoxins are currently being evaluated in clinical trials (7). The secondmajor approach for specific cancer immunotherapy employs the cellulararm of the immune system, mainly the CD8+ cytotoxic T-lymphocytes. Twomajor strategies are currently being used to increase the anti-tumoreffectiveness of the cellular arm of the immune system: (i) activeimmunization of patients with peptides known to be recognized byT-lymphocytes, and (ii) adoptive transfer therapies that enable theselection, activation, and expansion of highly reactive T-cellsubpopulations with improved anti-tumor potency. In the first approach,MHC-restricted peptides derived from recently identified tumorassociated antigens (such as gp100, the MAGE group, NY-ESO-1) are usedto vaccinate patients. These tumor specific antigen-derived peptides arehighly specific due to their exclusive expression in specific tissues(8-11). The second strategy, adoptive cell transfer, has recently shownimpressive results in metastatic melanoma patients in which highlyselected, tumor-reactive T-cells against different over-expressedself-derived differentiation antigens were isolated, expended ex-vivoand reintroduced to the patients. In this approach, a persistent clonalrepopulation of T-cells, proliferation in vivo, functional activity, andtrafficking to tumor sites were demonstrated (12-14).

A new immunotherapeutic approach recently presented takes advantage oftwo well-established areas: (i) the known effectiveness of CD8+cytotoxic T-lymphocytes in the elimination of cells presenting highlyimmunogenic MHC/peptide complexes, and (ii) the tumor-specific cellsurface antigens targeting via recombinant fragments of antibodies,mainly single chain Fv fragments (scFvs). This approach utilizes arecombinant fusion protein composed of two functionally distinctentities: (i) a single-chain MHC class I molecule that carries a highlyimmunogenic tumor or viral-derived peptide, and (ii) a tumor-specific,high-affinity scFv fragment (15). Several groups have previously shownthat a biotinylated MHC peptide multimerized on streptavidin ormonomeric HLA-A2/influenza (Flu) matrix peptide complexes coupled viachemical conjugation to tumor-specific antibodies could induce in vitroT-lymphocyte-mediated lysis of coated tumor cells (16-20). However,these approaches utilize chemical conjugation and use whole antibodiesor larger fragments, e.g. Fab fragments. However, production andhomogeneity owing to the coupling strategy as well as tumor penetrationcapability are limited due to the large size of such molecules. Lev etal. describe a genetic fusion created between a single-chain recombinantHLA-A2 and tumor specific scFvs. These fusions were shown to befunctional in vitro and in vivo, being able to specifically induceT-lymphocyte mediated in vitro and in vivo lysis of target-coated tumorcells (15). The stability of the new chimeric molecule is highlydependent on the presence of the peptide in the MHC groove. Therefore,dissociation of the peptide from the scHLA-A2 domain of the chimericmolecule can impair its stability. Oved et al. addressed this problem byconstructing new chimeric molecules in which the peptide is connected tothe scHLA-A2/scFv construct via a short linker. This new fusion proteinwas tested for its in vitro biochemical and biological activity (21).

There is a widely recognized need for a new fusion protein that canmaintain its dual activity: bind tumor target cells through the scFvmoiety as well as mediate potent, effective and specific cytotoxicitythrough the recruitment of CD8+ T-cells whose specificity is governed bythe covalently linked HLA-A2-restricted peptide.

The MHC class I-restricted CD8+ cytotoxic T-cell (CTL) effector arm ofthe adaptive immune response is best equipped to recognize tumor cellsas foreign and initiate the cascade of events resulting in tumordestruction. However, tumors have developed sophisticated strategies toescape immune effector mechanisms, of which the best-studied is thedownregulation of MHC class I molecules which present the antigensrecognized by CTLs.

To overcome the limitation of previous approaches and develop newapproaches for immunotherapy, a recombinant molecule was constructed inwhich a single-chain MHC is specifically targeted to tumor cells throughits fusion to cancer specific-recombinant antibody fragments or a ligandthat binds to receptors expressed by tumor cells.

SUMMARY OF THE INVENTION

This invention provides a fusion protein comprising consecutive aminoacids which, beginning at the amino terminus of the protein, correspondto consecutive amino acids present in (i) a cytomegalovirus humanMHC-restricted peptide, (ii) a first peptide linker, (iii) a human β-2microglobulin, (iv) a second peptide linker, (v) a HLA-A2 chain of ahuman MHC class I molecule, (vi) a third peptide linker, (vii) avariable region from a heavy chain of a scFv fragment of an antibody,and (viii) a variable region from a light chain of such scFv fragment,wherein the consecutive amino acids which correspond to (vii) and (viii)are bound together directly by a peptide bond or by consecutive aminoacids which correspond to a fourth peptide linker and the scFv fragmentis derived from an antibody which specifically binds to mesothelin.

This invention also provides compositions comprising the fusion proteinand a carrier.

This invention further provides a nucleic acid construct encoding afusion protein comprising consecutive amino acids which, beginning atthe amino terminus of the protein, correspond to consecutive amino acidspresent in (i) a cytomegalovirus human MHC-restricted peptide, (ii) afirst peptide linker, (iii) a human β-2 microglobulin, (iv) a secondpeptide linker, (v) a HLA-A2 chain of a human MHC class I molecule, (vi)a third peptide linker, (vii) a variable region from a heavy chain of ascFv fragment of an antibody, and (viii) a variable region from a lightchain of such scFv fragment, wherein the consecutive amino acids whichcorrespond to (vii) and (viii) are bound together directly by a peptidebond or by consecutive amino acids which correspond to a fourth peptidelinker and the scFv fragment is derived from an antibody whichspecifically binds to mesothelin.

This invention still further provides an isolated preparation ofbacterially-expressed inclusion bodies comprising over 30 percent byweight of a fusion protein in accordance with the invention.

This invention also provides a process for producing a fusion proteincomprising culturing a transformed cell comprising the fusion protein,so that the fusion protein is expressed, and recovering the fusionprotein so expressed.

This invention further provides a method of selectively killing a tumorcell which comprises contacting the cell with the fusion protein of theinvention in an amount effective to initiate a CTL-mediated immuneresponse against the tumor cell so as to thereby kill the tumor cell.

Finally, this invention further provides a method of treating a tumorcell which expresses mesothelin on its surface, which comprisescontacting the tumor cell with the fusion protein according to theinvention in an amount effective to initiate a CTL-mediated immuneresponse against the tumor cell so as to thereby treat the tumor cell.

As an exemplary molecule of the present invention, a single-chain MHCmolecule composed of β2 microglobulin fused to the α1, α2 and α3 domainsof HLA-A2 via a short peptide linker (15 amino acids) was fused to thescFv SS1 which targets mesothelin. To construct a fusion protein withcovalently linked peptide a 9 amino acids peptide derived from the CMVpp65 protein NLVPMVATV(SEQ ID NO:4) was fused to the N-terminus of thescHLA-A2/SS1(scFv) fusion protein via a 20 amino acid linkerGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6). The fusion protein was expressed inE. coli and functional molecules were produced by in vitro refolding inthe presence of CMV/scHLA-A2/SS1(scFv). Flow cytometry studies revealedthe ability to decorate antigen-positive, HLA-A2-negative human tumorcells with HLA-A2-peptide complexes in a manner that was entirelydependent upon the specificity of the targeting antibody fragment. Mostimportantly, CMV/scHLA-A2/SS1 (scFv)-mediated coating of target tumorcells made them susceptible for efficient and specificHLA-A2-restricted, CMV peptide-specific CTL-mediated lysis. Theseresults demonstrate that antibody-guided tumor antigen-specifictargeting of MHC-peptide complexes on tumor cells can render themsusceptible to, and potentiate, CTL killing. This novel approach nowopens the way for the development of new immunotherapeutic strategiesbased on antibody targeting of natural cognate MHC ligands and CTL-basedcytotoxic mechanisms.

In connection with the present invention, a novel strategy was developedto re-target class I MHC-peptide complexes on the surface of tumor cellsin a way that is independent of the extent of class I MHC expression bythe target tumor cells. To this end, in one embodiment of the presentinvention, a molecule with two arms was employed. One arm, the targetingmoiety, comprises tumor-specific recombinant fragments of antibodiesdirected to tumor or differentiation antigens which have been used formany years to target radioisotopes, toxins or drugs to cancer cells. Thesecond effector arm is a single-chain MHC molecule (scMHC) composed ofhuman β2-microglobulin linked to the three extracellular domains of theHLA-A2 heavy chain (24, 25, WO 01/72768). By connecting genes encodingthe two arms in a single recombinant gene and expressing the gene, thenew molecule is expressed efficiently in E. coli and produced, forexample, by in vitro refolding in the presence of HLA-A2-CMV peptides.This approach, as described herein, renders the target tumor cellssusceptible to lysis by cytotoxic T-cells regardless of their MHCexpression level and thus may be employed as a new approach topotentiate CTL-mediated anti-tumor immunity. This novel approach willlead to the development of a new class of recombinant therapeutic agentscapable of selective killing and elimination of tumor cells utilizingnatural cognate MHC ligands and CTL-based cytotoxic mechanisms.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A-1B

Schematic representation of scHLA-A2/SS1 (scFv) and apep(CMV)/scHLA-A2/SS1 (scFv) (Compound A).

FIG. 1A illustrates the C-Terminus of the scHLA-A2 fused to theN-terminus of SS1 (scFv) via a 4 amino acid linker. FIG. 1B illustratesthat the CMV pp65 peptide, i.e. NLVPMVATV (SEQ ID NO:4) was fused to theN-terminus of the scHLA-A2/SS1 (scFv) via a 20 amino acid linkerGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6).

FIG. 2

Nucleic acid sequence encoding Compound A (SEQ ID NO:1).

FIGS. 3A-3B

Expression and purification of Compound A.

FIG. 3A shows the SDS/PAGE analysis of isolated inclusion bodies. FIG.3B shows the SDS/PAGE analysis of Compound A after purification onion-exchange chromatography.

FIG. 4

Binding of Compound A to recombinant mesothelin.

Mesothelin was immobilized on immuno-plates and dose-dependent bindingof Compound A was monitored by conformation sensitive mAb W6 (33,34).

FIGS. 5A-5D

Binding of Compound A to mesothelin-expressing cells.

FIG. 5A-B demonstrates the flow cytometry analysis of the binding ofCompound A to mesothelin-positive HLA-A2-negative A431K5 cells andmesothelin-negative HLA-A2-negative A431 cells. FIG. 5A shows thebinding of the K1 mAb (31,32) to A431K5 cells, and FIG. 5B shows theabsence of binding of of the K1 mAb to A431 cells. FIG. 5C shows thebinding of Compound A to A431K5 cells, and FIG. 5D shows the absence ofbinding of Compound A to A431 cells. The binding was monitored usinganti-HLA-A2 specific antibody BB7.2 (35) and a FITC-labeled secondaryantibody.

FIGS. 6A-6B

Potentiation of CTL-mediated lysis of HLA-A2 negative tumor cells byCompound A. In FIG. 6A, the mesothelin-transfected A431K5 cells and theparental mesothelin-negative A431 cells were incubated with Compound A(10m) and CMV specific CTLs in a [S35]methionine release assay. FIG. 6Bdemonstrates dose-dependent activity of Compound A whenmesothelin-transfected A431K5 cells and the parental mesothelin-negativeA431 cells were incubated with different concentrations of Compound Aand CMV-specific CTLs in a [S35]methionine release assay.

FIG. 7

Schematic representation of the pep/scHLA-A2/SS1(scFv) (Compound B). InCompound B, the peptide NLVPMVATV (SEQ ID NO:4) was fused to theN-terminus of scHLA-A2/SS1 (scFv) via a 15 amino acid linkerGGGGSGGGGSGGGGS (SEQ ID NO:8).

FIG. 8

Nucleic acid sequence encoding Compound B (SEQ ID NO:22).

FIGS. 9A-9B

Expression and purification of Compound B.

FIG. 9A shows SDS/PAGE analysis of isolated inclusion bodies. FIG. 9Bshows SDS/PAGE analysis of Compound B after purification on ion-exchangechromatography.

FIGS. 10A-10F

Binding of Compound B to mesothelin-expressing cells.

FIGS. 10A-10F demonstrate the flow cytometry analysis of the binding ofCompound B to mesothelin-positive HLA-A2-negative A431K5 cells andmesothelin-negative HLA-A2-negative A431 cells. FIG. 10A shows thebinding of K1 mAb to A431K5 cells, and FIG. 10B shows the lack ofbinding of K1 mAb to A431 cells (B). FIG. 10C shows the binding ofCompound B to A431K5 cells, and FIG. 10D shows the lack of binding ofCompound B to A431 cells. FIG. 10E shows the comparison between thebinding of Compound A and Compound B to A431K5 cells, and FIG. 1OF showsthe lack of binding of Compound A and Compound B to A431 cells. Thebinding was monitored using anti-HLA-A2 specific antibody BB7.2 and aFITC-labeled secondary antibody.

FIGS. 11A-11B

Potentiation of CTL-mediated lysis of HLA-A2-negative tumor cells byCompound B.

In FIG. 11A, mesothelin-transfected A431K5 cells and the parentalmesothelin-negative A431 cells were incubated with Compound B (10 μg)and CMV-specific CTLs in a [S35]methionine release assay. FIG. 11Bdemonstrates dose-dependent activity of Compound B, whenmesothelin-transfected A431K5 cells and the parental mesothelin-negativeA431 cells were incubated with different concentrations of Compound Aand CMV-specific CTLs in a [S35]methionine release assay.

FIG. 12

Potentiation of CTL-mediated lysis of HLA-A2 negative tumor cells byCompound B and Compound A. Mesothelin-transfected A431K5 cells and theparental mesothelin-negative A431 cells were incubated with differentconcentrations of Compound B or Compound A and with CMV-specific CTLs ina [S35]methionine release assay. The figure shows results of incubationof Compound A with A431K5 cells, incubation of Compound B with A431K5cells, incubation of Compound A with A431 cells, and incubation ofCompound B with A431 cells.

FIGS. 13A-13B

Schematic representation of scHLA-A2/SS1 (scFv) and M1cov/scHLA-A2/SS1(scFv).

FIG. 13A shows the C-terminus of the scHLA-A2 fused to the N-terminus ofscFv via 4 amino acid linker. FIG. 13B shows the M158-66 peptide fusedto the N-terminus of the scHLA-A2/SS1(scFv) via a 15 amino acid linkerGGGGSGGGGSGGGGS (SEQ ID NO:8).

FIG. 14

Nucleic acid sequence encoding the M1cov/scHLA-A2/SS1 (scFv) fusionprotein (SEQ ID NO:23).

FIGS. 15A-15B

Expression and purification of the M1-cov/scHLA-A2/SS1 (scFv) fusionprotein.

FIG. 15A shows the SDS/PAGE analysis of isolated inclusion bodies. FIG.15B shows the SDS/PAGE analysis of M1-cov/scHLA-A2/SS1 (scFv) fusionprotein after purification on ion-exchange chromatography.

FIG. 16

Binding of the M1-cov/scHLA-A2/SS1 (scFv) fusion protein to recombinantMesothelin. Mesothelin was immobilized onto immuno-plates anddose-dependent binding of M1-cov/scHLA-A2/SS1 (scFv) was monitored byconformation sensitive mAb (W6).

FIGS. 17A-17D

Binding of M1-cov/scHLA-A2/SS1 (scFv) fusion protein to Mesothelinexpressing cells. FIGS. 17A-17D show flow cytometry analysis of thebinding of M1-cov/scHLA-A2/SS1 (scFv) to mesothelin-positiveHLA-A2-negative A431K5 cells and mesothelin-negative HLA-A2-negativeA431 cells. FIG. 17A shows the binding of K1 mAb to A431K5 cells, andFIG. 17B shows the absence of binding of K1 mAb to A431 cells. FIG. 17Cshows the binding of M1-cov/scHLA-A2/SS1 (scFv) fusion protein to A431K5cells, and FIG. 17D shows the absence of binding of M1-cov/scHLA-A2/SS1(scFv) fusion protein to A431 cells. The binding was monitored usinganti-HLA-A2 specific antibody BB7.2 and a FITC-labeled secondaryantibody.

FIG. 18

Potentiation of CTL-mediated lysis of HLA-A2-negative tumor cells byM1-cov/scHLA-A2/SS1 (scFv) fusion protein. Mesothelin-transfected A431K5cells and the parental mesothelin-negative A431 cells were incubatedwith different concentration of M1-cov/scHLA-A2/SS1 (scFv) and with M1specific HLA-A2-restricted CTLs in a [S35]methionine release assay.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

This invention provides a fusion protein comprising consecutive aminoacids which, beginning at the amino terminus of the protein, correspondto consecutive amino acids present in (i) a cytomegalovirus humanMHC-restricted peptide, (ii) a first peptide linker, (iii) a human β-2microglobulin, (iv) a second peptide linker, (v) a HLA-A2 chain of ahuman MHC class I molecule, (vi) a third peptide linker, (vii) avariable region from a heavy chain of a scFv fragment of an antibody,and (viii) a variable region from a light chain of such scFv fragment,wherein the consecutive amino acids which correspond to (vii) and (viii)are bound together directly by a peptide bond or by consecutive aminoacids which correspond to a fourth peptide linker and the scFv fragmentis derived from an antibody which specifically binds to mesothelin. Inone embodiment, the first peptide linker has the amino acid sequenceGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6). In another embodiment, the secondpeptide linker has the amino acid sequence GGGGSGGGGSGGGGS (SEQ IDNO:8). In another embodiment, the third peptide linker has the aminoacid sequence ASGG (SEQ ID NO:10). In another embodiment, the fourthpeptide linker has the amino acid sequence GVGGSGGGGSGGGGS (SEQ IDNO:19). In another embodiment, the cytomegalovirus human MHC-restrictedpeptide has the amino acid sequence NLVPMVATV (SEQ ID NO:4).

As used herein, “first peptide linker”, “second peptide linker” and“fourth peptide linker” refer to peptides composed of a monomericpeptide whose amino acid sequence is GXGGS (SEQ ID NO:20) or a multimerthereof, wherein X may be any amino acid. These peptide linkers may be amultimer of 2-10 of such monomeric peptide. In any such multimer, eachmonomeric peptide may be the same as or different from other monomericpeptide in the multimer depending on the identity of amino acid X. Inone embodiment, X in the monomeric peptide is the amino acid valine (V).In another embodiment, X in the monomeric peptide is the amino acidglycine (G). In presently preferred embodiments, the peptide linkercomprises a multimer of three or four monomeric peptides, particularly amultimer of three monomeric peptides in which the most N-terminal X isthe amino acid V, and the second and third X are the amino acid G.

In one embodiment, the sequence of the consecutive amino acidscorresponding to (vii), followed by the fourth peptide linker, followedby (viii) is set forth in SEQ ID NO:12.

In another embodiment, the consecutive amino acids of the fusionprotein, Compound A, have the amino acid sequence set forth in SEQ IDNO:2.

This invention also provides a composition comprising a fusion proteinin accordance with the invention and a carrier. In one embodiment, thefusion protein is present in the composition in a therapeuticallyeffective amount and the carrier is a pharmaceutically acceptablecarrier.

This invention also provides a nucleic acid construct encoding a fusionprotein comprising consecutive amino acids which, beginning at the aminoterminus of the protein, correspond to consecutive amino acids presentin (i) a cytomegalovirus human MHC-restricted peptide, (ii) a firstpeptide linker, (iii) a human β-2 microglobulin, (iv) a second peptidelinker, (v) a HLA-A2 chain of a human MHC class I molecule, (vi) a thirdpeptide linker, (vii) a variable region from a heavy chain of a scFvfragment of an antibody, and (viii) a variable region from a light chainof such scFv fragment, wherein the consecutive amino acids whichcorrespond to (vii) and (viii) are bound together directly by a peptidebond or by consecutive amino acids which correspond to a fourth peptidelinker and the scFv fragment is derived from an antibody whichspecifically binds to mesothelin. In one embodiment, the nucleic acidconstruct has the nucleic acid sequence set forth in SEQ ID NO:1.

This invention also provides a vector comprising the nucleic acidconstruct of the invention. Examples of such vectors are plasmids,viruses, phages, and the like.

This invention further provides an expression vector comprising thenucleic acid construct of the invention and a promoter operativelylinked thereto.

This invention also provides a transformed cell comprising a vectoraccording to the invention. The transformed cell may be a eukaryoticcell, e.g. one selected from the group consisting of a mammalian cell,an insect cell, a plant cell, a yeast cell and a protozoa cell.Alternatively, the transformed cell may be a bacterial cell.

This invention provides an isolated preparation of bacterially-expressedinclusion bodies comprising over 30 percent by weight of a fusionprotein according to the invention.

This invention also provides a process for producing a fusion proteincomprising culturing the transformed cell of the invention so that thefusion protein is expressed, and recovering the fusion protein soexpressed. In one embodiment, the recovery of the fusion proteincomprises subjecting the expressed fusion protein to size exclusionchromatography. In another embodiment, the fusion protein is expressedin inclusion bodies. In one embodiment, the process further comprisestreating the inclusion bodies so as to separate and refold the fusionprotein and thereby produce the fusion protein in active form. Inanother embodiment, treating of the inclusion bodies to separate thefusion protein therefrom comprises contacting the inclusion bodies witha denaturing agent.

As used herein, an “active form” of the fusion protein means a threedimensional conformation of the fusion protein which permits the fusionprotein to specifically bind to mesothelin when mesothelin is present onthe surface of a tumor cell.

This invention also provides a method of selectively killing a tumorcell, which comprises contacting the cell with the fusion protein of theinvention in an amount effective to initiate a CTL-mediated immuneresponse against the tumor cell so as to thereby kill the tumor cell. Inone embodiment, the tumor cell is in a patient and the contacting iseffected by administering the fusion protein to the patient.

This invention further provides a method of treating a tumor cell whichexpresses mesothelin on its surface, which comprises contacting thetumor cell with the fusion protein according to the invention in anamount effective to initiate a CTL-mediated immune response against thetumor cell so as to thereby treat the tumor cell. In one embodiment, thetumor cell is present in a solid tumor. In another embodiment, the solidtumor is a tumor associated with ovarian, lung, pancreatic or head/neckcancer, or mesothelioma.

The present invention provides (i) novel fusion proteins; (ii) processesof preparing same; (iii) nucleic acid constructs encoding same; and (iv)methods of using same for selective killing of cells, cancer cells inparticular.

The principles and operation of the present invention may be betterunderstood with reference to the figures and description set forthherein.

It is to be understood that the invention is not limited in itsapplication to the details set forth in the description or asexemplified. The invention encompasses other embodiments and is capableof being practiced or carried out in various ways. Also, it is to beunderstood that the phraseology and terminology employed herein is forthe purpose of description and should not be regarded as limiting.

Tumor progression is often associated with the secretion ofimmune-suppressive factors and/or the down-regulation of MHC class Iantigen-presentation functions (2). Even when a specific CTL response isdemonstrated in patients, this response is low because the anti-tumorCTL population is rare, very infrequent, and in some cases the CLTs arenot functional or anergic (26). Moreover, it is well-established thatthe number of MHC-peptide complexes on the surface of tumor cells thatpresent a particular tumor-associated peptide is low (27). Significantprogress toward developing vaccines that can stimulate an immuneresponse against tumors has involved the identification of the proteinantigens associated with a given tumor type and epitope mapping of tumorantigens for MHC class I and class II restricted binding motifs wereidentified and are currently being used in various vaccination programs(14, 11,8). MHC class I molecules presenting the appropriate peptidesare necessary to provide the specific signals for recognition andkilling by CTLs. However, the principal mechanism of tumor escape is theloss, downregulation or alteration of HLA profiles that may render thetarget cell unresponsive to CTL lysis, even if the cell expresses theappropriate tumor antigen.

The present invention provides a new approach to circumvent thisproblem. While reducing the present invention to practice,tumor-specific targeting of class I MHC-peptide complexes on tumor cellswas shown to be an effective and efficient strategy to renderHLA-A2-negative cells susceptible to lysis by relevant HLA-A2-restrictedCTLs. This new strategy of redirecting CTLs against tumor cells takesadvantage of the use of recombinant anti-mesothelin antibody fragmentand CMV ligand that can localize on malignant cells that express a tumorwith a relatively high degree of specificity.

The anti-mesothelin antibody targeting fragment and CMV ligand are fusedto a single-chain HLA-A2 molecule that can be folded efficiently andfunctionally.

The results presented herein provide a clear demonstration of theusefulness of the approach of the present invention to recruit activeCTLs for tumor cell killing via cancer-specific antibody or ligandguided targeting of scMHC-peptide complexes. These results pave the wayfor the development of a new immunotherapeutic approach based onnaturally occurring cellular immune responses which are redirectedagainst the tumor cells.

It will be appreciated that the fusion protein of the present inventionor portions thereof can be prepared by several ways, including solidphase protein synthesis. However, in the preferred embodiment of theinvention, at least major portions of the molecules, e.g., the scHLA-A2domain (with or without the CMV peptide) and the scFV domain aregenerated by translation of a respective nucleic acid construct orconstructs encoding the molecule.

Accordingly, one to three open reading frames are required to synthesizethe molecules of FIG. 1B via translation. These open reading frames canreside on a single, two or three nucleic acid molecules. Thus, forexample, a single nucleic acid construct can carry one, two or all threeopen reading frames. One to three cis-acting regulatory sequences can beused to control the expression of the one to three open reading frames.For example, a single cis-acting regulatory sequence can control theexpression of one, two or three open reading frames, in a cistrone-likemanner. In the alternative, three independent cis-acting regulatorysequences can be used to control the expression of the three openreading frames. Other combinations are also envisaged.

The open reading frames and the cis-acting regulatory sequences can becarried by one to three nucleic acid molecules. For example, each openreading frame and its cis-acting regulatory sequence are carried by adifferent nucleic acid molecule, or all of the open reading frames andtheir associated cis-acting regulatory sequences are carried by a singlenucleic acid molecule. Other combinations are also envisaged.

Expression of the fusion protein can be effected bytransformation/transfection and/or co-transformation/co-transfection ofa single cell or a plurality of cells with any of the nucleic acidmolecules, serving as transformation/transfection vectors (e.g., asplasmids, phages, phagemids or viruses).

It will be appreciated that the fusion protein whose amino acid sequenceis set forth in SEQ ID NO:2 and includes the N-terminal amino acidmethionine, likely represents the fusion protein as expressed in abacterial cell. Depending on the specific bacterial cell employed toexpress the fusion protein, the N-terminal methionine may be cleaved andremoved. Accordingly, it is contemplated that fusion proteins inaccordance with this invention encompass both those with, and thosewithout, a N-terminal methionine. In general, when a fusion protein inaccordance with the invention is expressed in a eukaryotic cell, itwould lack the N-terminal methionine. Therefore, it is to be appreciatedthat the amino acid sequence of expressed fusion proteins according tothe invention may include or not include such N-terminal methioninedepending on the type of cells in which the proteins are expressed.

Whenever and wherever used, the linker peptide is selected of an aminoacid sequence which is inherently flexible, such that the polypeptidesconnected thereby independently and natively fold following expressionthereof, thus facilitating the formation of a functional or activesingle chain (sc) human β2M/HLA complex, antibody targeting or humanβ2M/HLA -CMV restricted antigen complex.

Any of the nucleic acid constructs described herein comprise at leastone cis-acting regulatory sequence operably linked to the codingpolynucleotides therein. Preferably, the cis-acting regulatory sequenceis functional in bacteria. Alternatively, the cis-acting regulatorysequence is functional in yeast. Still alternatively, the cis-actingregulatory sequence is functional in animal cells. Yet alternatively,the cis acting regulatory sequence is functional in plant cells.

The cis-acting regulatory sequence can include a promoter sequence andadditional transcriptional or a translational enhancer sequences all ofwhich serve for facilitating the expression of the polynucleotides whenintroduced into a host cell. Specific examples of promoters aredescribed hereinbelow in context of various eukaryotic and prokaryoticexpression systems and in the examples section which follows.

It will be appreciated that a single cis-acting regulatory sequence canbe utilized in a nucleic acid construct to direct transcription of asingle transcript which includes one or more open reading frames. In thelater case, an internal ribosome entry site (IRES) can be utilized so asto allow translation of the internally positioned nucleic acid sequence.

Whenever co-expression of independent polypeptides in a single cell isof choice, the construct or constructs employed must be configured suchthat the levels of expression of the independent polypeptides areoptimized, so as to obtain highest proportions of the final product.

Preferably a promoter (being an example of a cis-acting regulatorysequence) utilized by the nucleic acid construct(s) of the presentinvention is a strong constitutive promoter such that high levels ofexpression are attained for the polynucleotides following host celltransformation.

It will be appreciated that high levels of expression can also beeffected by transforming the host cell with a high copy number of thenucleic acid construct(s), or by utilizing cis acting sequences whichstabilize the resultant transcript and as such decrease the degradationor “turn-over” of such a transcript.

As used herein, the phrase “transformed cell” describes a cell intowhich an exogenous nucleic acid sequence is introduced to thereby stablyor transiently genetically alter the host cell. It may occur undernatural or artificial conditions using various methods well known in theart some of which are described in detail hereinbelow in context withspecific examples of host cells.

The transformed host cell can be a eukaryotic cell, such as, forexample, a mammalian cell, an insect cell, a plant cell, a yeast celland a protozoa cell, or alternatively, the cell can be a bacterial cell.

When utilized for eukaryotic host cell expression, the nucleic acidconstruct(s) according to the present invention can be a shuttle vector,which can propagate both in E. coli (wherein the construct comprises anappropriate selectable marker and origin of replication) and becompatible for expression in eukaryotic host cells. The nucleic acidconstruct(s) according to the present invention can be, for example, aplasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or anartificial chromosome.

Suitable mammalian expression systems include, but are not limited to,pcDNA3, pcDNA3.1(+/−), pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto,pCMV/myc/cyto, pCR3.1, which are available from Invitrogen™ Corporation(Carlsbad, Calif. USA), pCI which is available from Promega™ Corporation(Madison Wis. USA), pBK-RSV and pBK-CMV which are available fromStratagene® (La Jolla, Calif. USA), pTRES which is available fromClontech® Laboratories, Inc. (Mountain View, Calif. USA), and theirderivatives.

Insect cell cultures can also be utilized to express the nucleic acidsequences of the present invention. Suitable insect expression systemsinclude, but are not limited to the baculovirus expression system andits derivatives which are commercially available from numerous supplierssuch as maxBac™ (Invitrogen™ Corporation, Carlsbad, Calif. USA) BacPak™(Clontech® Laboratories, Inc. Mountain View, Calif. USA), or Bac-to-Bac™(Invitrogen™/Gibco®, Carlsbad, Calif. USA).

Expression of the nucleic acid sequences of the present invention canalso be effected in plants cells. As used herein, the phrase “plantcell” can refer to plant protoplasts, cells of a plant tissue culture,cells of plant derived tissues or cells of whole plants.

There are various methods of introducing nucleic acid constructs intoplant cells. Such methods rely on either stable integration of thenucleic acid construct or a portion thereof into the genome of the plantcell, or on transient expression of the nucleic acid construct in whichcase these sequences are not stably integrated into the genome of theplant cell.

There are two principle methods of effecting stable genomic integrationof exogenous nucleic acid sequences such as those included within thenucleic acid construct of the present invention into plant cell genomes:

-   -   (i) Agrobacterium-mediated gene transfer: Klee et al. (1987)        Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell        Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular        Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L.        K., Academic Publishers, San Diego, Calif. (1989) p. 2-25;        Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, C.        J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.    -   (ii) direct DNA uptake: Paszkowski et al., in Cell Culture and        Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of        Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic        Publishers, San Diego, Calif. (1989) p. 52-68; including methods        for direct uptake of DNA into protoplasts, Toriyama, K. et        al. (1988) Bio/Technology 6:1072-1074. DNA uptake induced by        brief electric shock of plant cells: Zhang et al. Plant Cell        Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793.        DNA injection into plant cells or tissues by particle        bombardment, Klein et al. Bio/Technology (1988) 6:559-563;        McCabe et al. Bio/Technology (1988) 6:923-926; Sanford, Physiol.        Plant. (1990) 79:206-209; by the use of micropipette systems:        Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and        Spangenberg, Physiol. Plant. (1990) 79:213-217; or by the direct        incubation of DNA with germinating pollen, DeWet et al. in        Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P.        and Mantell, S. H. and Daniels, W. Longman, London, (1985) p.        197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure, see for example, Horsch et al. in PlantMolecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht(1988) p. 1-9. A supplementary approach employs the Agrobacteriumdelivery system in combination with vacuum infiltration. TheAgrobacterium system is especially viable in the creation of stablytransformed dicotyledenous plants.

There are various methods of direct DNA transfer into plant cells. Inelectroporation, protoplasts are briefly exposed to a strong electricfield. In microinjection, the DNA is mechanically injected directly intothe cells using very small micropipettes. In microparticle bombardment,the DNA is adsorbed on microprojectiles such as magnesium sulfatecrystals, tungsten particles or gold particles, and the microprojectilesare physically accelerated into cells or plant tissues. Direct DNAtransfer can also be utilized to transiently transform plant cells.

In any case suitable plant promoters which can be utilized for plantcell expression of the first and second nucleic acid sequences, include,but are not limited to CaMV 35S promoter, ubiquitin promoter, and otherstrong promoters which can express the nucleic acid sequences in aconstitutive or tissue specific manner.

Plant viruses can also be used as transformation vectors. Viruses thathave been shown to be useful for the transformation of plant cell hostsinclude CaV, TMV and BV. Transformation of plants using plant viruses isdescribed in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), JapanesePublished Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667(BV); and Gluzman, Y. et al., Communications in Molecular Biology: ViralVectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988).Pseudovirus particles for use in expressing foreign DNA in many hosts,including plants, is described in WO 87/06261.

Construction of plant RNA viruses for the introduction and expression ofnon-viral exogenous nucleic acid sequences in plants is demonstrated bythe above references as well as by Dawson, W. O. et al., Virology (1989)172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al.Science (1986) 231:1294-1297; and Takamatsu et al. FEBS Letters (1990)269:73-76.

When the virus is a DNA virus, the constructions can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe nucleic acid sequences described above. The virus can then beexcised from the plasmid. If the virus is a DNA virus, a bacterialorigin of replication can be attached to the viral DNA, which is thenreplicated by the bacteria. Transcription and translation of this DNAwill produce the coat protein which will encapsidate the viral DNA. Ifthe virus is an RNA virus, the virus is generally cloned as a cDNA andinserted into a plasmid. The plasmid is then used to make all of theconstructions. The RNA virus is then produced by transcribing the viralsequence of the plasmid and translation of the viral genes to producethe coat protein(s) which encapsidate the viral RNA.

Construction of plant RNA viruses for the introduction and expression inplants of non-viral exogenous nucleic acid sequences such as thoseincluded in the construct of the present invention is demonstrated bythe above references as well as in U.S. Pat. No. 5,316,931.

Yeast cells can also be utilized as host cells by the present invention.Numerous examples of yeast expression vectors suitable for expression ofthe nucleic acid sequences of the present invention in yeast are knownin the art and are commercially available. Such vectors are usuallyintroduced in a yeast host cell via chemical or electroporationtransformation methods well known in the art. Commercially availablesystems include, for example, the pYES™ (Invitrogen™ Corporation,Carlsbad Calif., USA) or the YEX™ (Clontech® Laboratories, MountainView, Calif. USA) expression systems.

It will be appreciated that when expressed in eukaryotic expressionsystems such as those described above, the nucleic acid constructpreferably includes a signal peptide encoding sequence such that thepolypeptides produced from the first and second nucleic acid sequencesare directed via the attached signal peptide into secretion pathways.For example, in mammalian, insect and yeast host cells, the expressedpolypeptides can be secreted to the growth medium, while in plantexpression systems the polypeptides can be secreted into the apoplast,or directed into a subcellular organelle.

A bacterial host can be transformed with the nucleic acid sequence viatransformation methods well known in the art, including for example,chemical transformation (e.g., CaCl2) or electroporation.

Numerous examples of bacterial expression systems which can be utilizedto express the nucleic acid sequences of the present invention are knownin the art. Commercially available bacterial expression systems include,but are not limited to, the pET™ expression system (Novagen®, EMBBiosciences, San Diego, Calif. USA), pSE™ expression system (Invitrogen™Corporation, Carlsbad Calif., USA) or the pGEX™ expression system(Amersham Biosciences, Piscataway, N.J. USA).

As is further described in the Experimental Details section whichfollows, bacterial expression is particularly advantageous since theexpressed polypeptides form substantially pure inclusion bodies readilyamenable to recovery and purification of the expressed polypeptide.

Thus, this invention provides a preparation of bacterial-expressedinclusion bodies which are composed of over 30%, preferably over 50%,more preferably over 75%, most preferably over 90% by weight of thefusion protein or a mixture of fusion proteins of the present invention.The isolation of such inclusion bodies and the purification of thefusion protein(s) therefrom are described in detail in the ExperimentalDetails section which follows. Bacterial expression of the fusionprotein(s) can provide high quantities of pure and active forms offusion proteins.

As is further described in the Experimental Details section whichfollows, the expressed fusion proteins form substantially pure inclusionbodies which are readily isolated via fractionation techniques wellknown in the art and purified via for example denaturing-renaturingsteps.

The fusion proteins of the invention may be renatured and refolded inthe presence of a MHC-restricted peptide, which is either linked to,co-expressed with or mixed with other polypeptides of the invention andbeing capable of binding the single chain MHC class I polypeptide. As isfurther described in the examples section, this enables to generate asubstantially pure MHC class I-antigenic peptide complex which canfurther be purified via size exclusion chromatography.

It will be appreciated that the CMV peptide used for refolding can beco-expressed along with (as an independent peptide) or be fused to thescHLA-A2 chain of the MHC Class I molecule in the bacteria. In such acase the expressed fusion protein and peptide co-form inclusion bodieswhich can be isolated and utilized for MHC class I-antigenic peptidecomplex formation.

The following section provides specific examples for each of the variousaspects of the invention described herein. These examples should not beregarded as limiting in any way, as the invention can be practiced insimilar, yet somewhat different ways. These examples, however, teach oneof ordinary skills in the art how to practice various alternatives andembodiments of the invention.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R.I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

EXPERIMENTAL DETAILS

Materials and Methods:

Cloning of Compound A

The scHLA-A2/SS1 (scFv) was constructed as previously described bylinking the C-terminus of scHLA-A2 to the N-terminus of the SS1 scFv viaa short linker ASGG (SEQ ID NO:4) (15). To construct the scHLA-A2/SS1(scFv) with covalently bound MHC-restricted peptide, the MHC-restrictedpeptide was fused with the peptide linker GGGGSGGGGSGGGGSGGGGS (SEQ IDNO:6) to the N-terminus of the scHLA-A2/SS1 (scFv) molecule by a PCRoverlap extension reaction with the primers:5′M1-5′GGAAGCGTTGGCGCATATGGGCATTCTGGGCTTCGTGTTTACCCTGGGCGGAGGAGGATCCGGTGGCGGAGGTTCAGGAGGCGGTGGATCGATCCAGCGTACTCCAAAG3′(SEQ ID NO: 13) and 3′VLscS S1-5′GCAGTAAGGAATTCTCATTATTTTATTTCCAACTTTGT3′ (SEQ ID NO: 14). In the 5′M1 primer a silence mutation was inserted at the linker sequence, thischange in sequence creates a BamH1 restriction site.

The PCR products were sub-cloned to TA cloning vector (pGEM-T EasyVector, Promega™ Corporation, Madison, Wis. USA) and subsequently to aT7 promoter-based expression vector (PRB) using the NdeI and EcoRIrestriction sites.

To generate Compound A, M1/scHLA-A2/SS1 (scFv) was used as a templatefor ligation with dsDNA primer. The M1/scHLA-A2/SS1 (scFv) (in PRBplasmid) was digested with NdeI and BamHI, and the plasmid fraction wasligated to dsDNA primer containing the CMV peptide sequence and theextension of the linker sequence 5′CMVcovLL(cassette):5′TATGAACCTGGTGCCGATGGTCGCGACCGTTGGAGGTGGCGGTTCTGGCGGAGGAG-3′ (SEQ ID NO: 15) and 3′CMVcovLL(cassette):5′GATC CTCCTCCGCCAGAACCGCCACCTCCAACGGTCGCGACCATCGGCACCAGGTTCA3′(SEQ ID NO:16). The annealing of the primers (5′CMVcovLL (cassette)and 3′CMVcovLL (cassette)) was performed by incubating the primers at95° C. for 2 min followed by 1 h incubation at room temperature. Theligation product was transformed to E-coli DH5α for plasmidamplification. Plasmid was purified by QIAGEN® Miniprep™, DNA isolationkit (Qiagen®, Inc., Valencia, Calif. USA)and samples were set forsequence analysis.

Expression Refolding and Purification of Compound A

Compound A was expressed in E-coli LB21 (λDE3) cells (Novagen®, Madison,Wis. USA) as inclusion bodies. Compound A construct was transformed toE-coli cells by heat shock, cells were plated on LBAMP plates andincubated over night at 37° C. Colonies were transferred to rich medium(super broth) supplemented with glucose, MgSO4, AMP and salts. The cellswere grown to DO=2 (600 nm) at 37° C., induced with IPTG (finalconcentration 1 mM) and incubated for an additional 3 h at 37° C.

Inclusion bodies were purified from cell pellet by cell disruption with0.2 mg/ml of lysozyme followed by the addition of 2.5% Triton® X-100(Octylphenolpoly[ethyleneglycolether]X, Roche Diagnostics GmbH, RocheApplied Science, Mannheim, Germany) and 0.5M NaCl. The pellets of theinclusion bodies were collected by centrifugation (13,000 rpm, 60 min at4° C.) and washed three times with 50 mM Tris buffer pH 7.4 containing20 mM EDTA. The isolated and purified inclusion bodies were solubilizedin 6M Guanidine HCl pH 7.4, followed by reduction with 65-mM DTE.Solubilized and reduced inclusion bodies were refolded by a 1:100dilution into a redox-shuffling buffer system containing 0.1-M Tris,0.001M EDTA, 0.5-M Arginine, and 0.09-mM Oxidized Glutathione, pH 9, andincubation at 10° C. for 24 h. After having been refolded, the proteinwas dialyzed against 150-mM Urea, 20-mM Tris, pH 8, followed bypurification of the soluble Compound A by ionexchange chromatography ona Q-Sepharose® column (7.5 mm I.D 60 cm) (Sigma-Aldrich, Inc., St.Louis, Mo. USA), applying a salt (NaCl) gradient. Peak fractionscontaining Compound A were then subjected to buffer exchange with PBS.

Cloning of Compound B

The scHLA-A2/SS1 (scFv) was constructed as previously described bylinking the C-terminus of scHLA-A2 to the N-terminus of the SS1 scFv viaa short linker ASGG (SEQ ID NO:15). To construct the scHLA-A2/SS1 (scFv)with covalently bound MHC-restricted peptide, the MHC-restricted peptideand the peptide linker GGGGSGGGGSGGGGS (SEQ ID NO:8) were fused to theN-terminus of the scHLA-A2/SS1(scFv) molecule by a PCR overlap extensionreaction with the primers 5′-Nde-209B2M:5′GGAAGCGTTGGCGCATATGATCATGGACCAGGTTCCGTTCTCTGTTGGCGAGGAGGGTCCGGTGGCGGAGGTTCAGGAGGCGGTGGATCGATCCAGCGTACTCCAAAG3′(SEQ ID NO: 17) And the 3′VLscSS1-5′GCAGTAAGGAATTCTCAT TATTTTATTTCCAACTTTGT3′(SEQ ID NO:18). 209cov/scHLA-A2/SS1(scFv) molecule was used as a template for the construction of CompoundB. In this molecule the CMV peptide NLVPMVATV (SEQ ID NO: 4) wasintroduced into the 209cov/scHLA-A2/SS1 (scFv) sequence (exchanging the209 peptide) by PCR reaction using the primers 5′GGAAGCGTTGGCGCATATGGGCATTCTGGGCTTCGTGTTTACCCTGGGCGAGGAGGATCCGGTGGCGGAGGTTCAGGAGGCGGTGGATCGATCCAGCGTACTCCAAAG3′(SEQ ID NO: 17) and the3′VLscSS15′GCAGTAAGGAATTCTCATTATTTTAT TTCCAACTTTGT3′(SEQ ID NO: 18).

The expression and purification protocols of Compound B were identicalto the expression and purification protocols of Compound A.

All the methods used to analyze the biochemical and biologicalproperties of Compound B were identical to the methods used to analyzethe activity of Compound A.

Construction of M1-COV/scHLA-A2/SS1 (scFv)

To construct the M1-cov/scHLA-A2/SS1 (scFv) fusion protein the MI 58-66peptide was fused to the N-terminus of scHLA-A2/SS1 (scFv) fusionprotein through a short 15 amino acid linker by overlapping PCR reactionwith the 5′M1-linker primer:5′GGAAGCGTTGGCGCATATGGGCATTCTGGGCTTCGTGTTTACCCTGGGCGGAGGAGGATCCGGTGGCGGAGGTTCAGGAGGCGGTGGATCGATCCAGCGTACTC CAAAG3′(SEQ ID NO:13) and the 3′VLscSS1-5′GCAGTAAGGAATTCTCAT TATTTTATTTCCAACTTTGT3′(SEQ IDNO: 14). PRB plasmid was used as a template containing the scHLA-A2/SS1(scFv) sequence. The expression and purification protocols of theM1-cov/scHLA-A2/SS1 (scFv) fusion protein were identical to theexpression and purification protocols of Compound A. All the methodsused to analyse the biochemical and biological properties of theM1-cov/scHLA-A2/SS1 (scFv) fusion protein were identical to the methodsused to analyse the activity of Compound A.

Flow Cytometry

Cells were incubated with Compound A (60 min at 4° C. in 100 μl, 10μg/ml), washed and incubated with the anti-HLA-A2 MAb BB7.2 (60 min at4° C., 10 μl/ml). The cells were washed and incubated with anti-mouseFITC (60 min at 4° C., 10 μl/ml) that served as a secondary antibody.The cells were subsequently washed and analyzed by a FACS caliber flowcytometer (Becton-Dickinson, San Jose, Calif. USA).

Enzyme Linked Immunosorbent Assay

Immunoplates (Falcon®, Becton-Dickinson Labware, Franklin Lakes, N.J.USA) were coated with 10 μg/ml of purified bacterially producedrecombinant mesothelin (O/N at 4° C.). The plates were blocked with PBScontaining 2% skim milk and then incubated with various concentrationsof Compound A (60 min at RT) and washed three times with PBS. Bindingwas detected using the anti-HLA-conformational-dependent antibody W6/32(60 min, RT, 1 μg/ml), plates were washed three times with PBS andincubated with anti-mouse IgG-peroxidase (60 min, RT, 1 μg/ml). Thereaction was developed using TMB (DAKO) and terminated by the additionof 50 μl H2SO4 2N. Anti-mesothelin antibody (K1) was used as a positivecontrol. The immunoplates were analyzed by ELISA reader using 450 nmfilter (Anthos 2001™, Anthos Labtech, Salzburg, Austria).

Cytotoxicity Assays

Cytotoxicity was determined by S35-methionine release assays. Targetcells were cultured in culture plates in RPMI 10% FCS Methionine freefor 2 h, followed by incubation overnight with 15 μCi/ml ofS35methionine (NEN). The target cells were harvested by trypsinizationand washed twice with 40 ml RPMI 10% FCS. The target cells were platedin 96-well plates (5·103 cells per well) in RMPI+10% FCS and incubatedovernight at 37° C., 5% CO2. Target cells were incubated with differentconcentrations of Compound A fusion proteins for 2 h, effector CTL cellswere added at different target: effector ratios and the plates wereincubated for 8-12 h at 37° C., 5% CO2. Following incubation,S35-methionine release from target cells was measured in a 25 μl sampleof the culture supernatant. All assays were performed in triplicate,lysis was calculated directly: ([experimental release−spontaneousrelease]/[maximum release−spontaneous release])·100. Spontaneous releasewas measured as S35 methionine released from target cells in the absenceof effector cells, and maximum release was measured as S35-methioninereleased from target cells lyzed by 0.05M NaOH.

Cell Lines

A431 and A431K5 cells (epidermoid carcinoma) were maintained in RPMImedium containing 10% FCS, L-glutamine and penicillin/streptomycin. TheA431K5 cell line is a human epidermoid carcinoma A431 cell line stablytransfected with Mesothelin, the transfected cells were maintained with700 μg/ml G418 (Gibco-BRL®, Invitrogen™ Inc., Carlsbad, Calif. USA).

CTL's with specificity for CMV pp65 epitope (NLVPMVATV (SEQ ID NO:4))were kindly provided by Dr Ditmar Zehn (Charitee, Berlin). The CTL'swere expanded by incubation with peptide pulsed, radiated (4000rad)PBMC's from a healthy HLA-A2 positive donor and were maintained in AIMVmedium+8.9% FCS+50 μM-2-mercaptoethanol +penicillin/streptomycin 1·105U/L.

Results:

Construction of Compound A

A construct encoding a single-chain MHC molecule composed of the β2microglobulin gene fused to the α1, α2 and α3 of the HLA-A2 gene via ashort peptide linker (15 amino acids) was fused to the scFv SS1 whichtargets mesothelin (FIG. 1A). This construct was analyzed in detail forits biochemical and biological activity and was found to be functionalin-vitro and in-vivo (15). To construct a fusion protein with covalentlylinked peptide a 9 amino acids peptide derived from the CMV pp65 protein(NLVPMVATV) (SEQ ID NO:4) was fused to the N-terminus of thescHLA-A2/SS1(scFv) fusion protein via 20 amino acids linkerGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6) (FIG. 1B). Compound A was constructedin two steps: First a covalent fusion protein termedM1/scHLA-A2/SS1(scFv) was constructed by overlap extension PCR. In thisconstruct the influenza M158-66 peptide GILGFVFTL (SEQ ID NO:21) and a15 amino acid linker were fused to the N-terminus of the scHLA-A2/SS1(scFv) fusion protein. In this construct, a new, unique restriction site(BamHI) was inserted to the linker sequence by a silent mutation. In thesecond step PRB plasmid containing the M1/scHLA-A2/SS1 (scFv) fullsequence was digested with NdeI and BamHI restriction enzymes. Thisdigestion produced two fragments. One fragment contains the peptide andpart of the linker sequence, and the second fragment contains theplasmid, part of the linker and the scHLA-A2/SS1 (scFv) sequence. Thefragment which contains the plasmid, part of the linker and thescHLA-A2/SS1 (scFv) sequence was then ligated to dsDNA primer that codesthe CMV pp65 peptide sequence and an extension of the linker sequence(FIG. 1B). The new plasmid was transformed to E-coli DH5α cells andpositive colonies were sent to DNA sequencing (FIG. 2).

Expression and Purification of Compound A

Compound A was expressed in E. coli BL21 cells and, upon induction withisopropyl β-D-thiogalactoside, large amounts of recombinant proteinaccumulated in intracellular inclusion bodies. SDS/PAGE analysis ofisolated and purified inclusion bodies revealed that Compound A with thecorrect size constituted 80-90% of the total inclusion bodies mass (FIG.3A). The isolated solubilized inclusion bodies were reduced and refoldedin-vitro in a redox-shuffling buffer. Monomeric soluble fusion proteins(Compound A) were purified by ion-exchange chromatography onQ-sepharose®. SDS/PAGE analysis of Compound A revealed a highly purifiedmonomeric molecule with the expected size of 72 KDa (FIG. 3B).

Biological Activity of the Compound A ELISA

To test the binding ability of purified Compound A to its targetantigen, the recombinant mesothelin was immobilized to immunoplates. Thebinding of Compound A was monitored by using conformation sensitive mAbW6/32, this antibody recognizes MHC molecules that are folded correctlywith a peptide in its groove. As shown in FIG. 4, the binding ofCompound A to recombinant mesothelin was dose-dependent. This suggeststhat the two functional domains of Compound A, the scFv (SS1) domain andthe peptide/scHLA-A2 domain are folded correctly. Moreover, the scFv(SS1) domain of the fusion protein is in active form and canspecifically bind mesothelin.

Flow Cytometry Analysis (FACS)

To test the binding ability of Compound A to mesothelin-expressing celllines, FACS analysis was made. As a model, target cells that are HLA-A2negative were used, thus the reactivity of an anti-HLA-A2 mAb can beused to measure the binding of Compound A to cells that expressmesothelin on their surface. This model of mesothelin-positive,HLA-A2-negative cells represents the extreme case in which the tumorcells lose its HLA expression. Therefore for the FACS analysis, HLA-A2negative A431K5 cells were used, which are human epidermoid carcinomaA431 cells that were stably transfected with mesothelin. The parentalA431 human epidermoid carcinoma cells which are mesothelin-negative andHLA-A2-negative are used as negative control. The binding of Compound Ato the target cells was monitored with anti-HLA-A2 mAb BB7.2 as primaryantibody followed by a FITC labeled secondary antibody. A mesothelinanti-mAb K1 was used to test the expression levels of mesothelin. Asshown in FIG. 5A, A431K5 cells express high levels of mesothelin,whereas the parental A431 cells do not express the target antigen. Thecell lines A431 and A431K5 were also tested for the expression of HLA-A2using HLA-A2 specific antibody (BB7.2), both cell lines were HLA-A2negative. However, when A431K5 cells were pre-incubated with Compound A,they were positively stained with the HLA-A2 specific antibody BB7.2(FIG. 5B). Antigen-negative A413 cells were not affected. The specificbinding of Compound A to A431K5 but not to A431 cells further indicatesthat the binding is exclusively depended on the interaction of thetargeting scFv domain of the fusion with mesothelin and that the fusionprotein can bind its target antigen as natively expressed on the surfaceof cells.

Cytotoxicity Assay

To test the ability of Compound A to mediate the killing ofHLA-A2-negative mesothelin-positive cells by HLA-A2-restricted CMV pp65NLVPMVATV (SEQ ID NO:4) specific CTLs, S35-Methionine release assay wasperformed using HLA-A2-negative mesothelin-transfected A431K5 cells, andthe HLA-A2-negative mesothelin-negative A431 parental cells. Todetermine the killing potential of the CMV specific CTLs, cytotoxicityassay was performed using HLA-A2-positive JY cells that wereradiolabeled with MetS35 and laded with the CMV peptide NLVPMVATV (SEQID NO:4). The average specific killing of the JY cells by the CMVspecific CTLs was 47% with an E:T ratio of 10:1 (data not shown). Asshown in FIG. 6A, Compound A effectively mediated the killing of theA431K5 cells (mesothelin-positive HLA-A2-negative). Specific killingcould reach 66% in comparison to peptide-loaded JY cells. Thus, killingwith the fusion protein was even more efficient compared topeptide-pulsed antigen presenting cells which represent optimal targets.However, when the target A431K5 cells were incubated with the CMVspecific CTLs alone without preincubation with Compound A or when thetarget cells were A431 mesothelin-negative cells with or withoutpreincubation with Compound A, no cytotoxic activity was observed. Next,a titration experiment was performed to determine the potency ofCompound A, as shown in FIG. 6B, the killing of mesothelin-positiveA431K5 cells was dose-dependent with an IC50 of 0.5-1 μg/ml.

These results indicate that the killing of mesothelin-positiveHLA-A2-negative A431K5 cell was specific and controlled by therecognition of mesothelin by the targeting domain of Compound A(scFv/SS1) and the specificity of the CMV CTLs to the peptide/scHLA-A2domain.

Biological Activity of Compound B Flow Cytometry Analysis (FACS)

To test the binding ability of Compound B to mesothelin-expressing celllines a flow cytometry analysis was used. As a model, target cells thatare HLA-A2 negative were used, thus, the reactivity of the anti-HLA-A2mAb will indicate the binding of Compound B to the cell surface antigen.A431K5 cells which are human epidermoid carcinoma A431 cells that werestably transfected with mesothelin and are HLA-A2 negative. As controlsthe parental A431 human epidermoid carcinoma cells were used which aremesothelin-negative and HLA-A2 negative. The binding of Compound B tothe target cells was monitored by anti-HLA-A2 mAb BB7.2 as primaryantibody followed by a FITC labeled secondary antibody. To test theexpression levels of mesothelin and as positive control a commercialanti-mesothelin mAb K1 was used. As shown in FIG. 10A-10B A431K5 cellsexpress high levels of mesothelin, whereas the parental A431 cells donot express mesothelin. The cell lines A431 and A431K5 were also testedfor their expression of HLA-A2 using HLA-A2 specific anti body (BB7.2),both cell lines were HLA-A2 negative. However, when A431K5 cells werepre-incubated with Compound B, they were positively stained with theHLA-A2 specific antibody BB7.2 whereas control A431 cells were notstained (FIG. 10 C-10D). The specific binding of Compound B to A431K5cells but not to A431 cells indicate that binding is exclusivelydepended on the interaction of the targeting scFv domain withmesothelin.

To analyze the binding of Compound B in comparison to the Compound Afusion, a FACS analysis was performed using both molecules in similarconditions and concentrations. As shown in FIGS. 10E-10F only themesothelin-positive cells A431K5 were positively stained withHLA-A2-specific Ab when pre-incubated with both fusion proteins,however, Compound A exhibited better binding.

Cytotoxicity Assay

To test the ability of Compound B to mediate the killing ofHLA-A2-negative mesothelin-positive cells by HLA-A2-restricted CMVNLVPMVATV (SEQ ID NO:4) specific CTLs, performed S35-Methionine releaseassay using HLA-A2-negative mesothelin-transfected A431K5 cells wasperformed, and the HLA-A2-negative mesothelin-negative A431 parentalcells. To determine the killing potential of the CMV-specific CTLscytotoxicity assay was performed, using HLA-A2-positive JY cells thatwere radiolabeled with MetS35 and laded with the CMV peptide NLVPMVATV(SEQ ID NO: 4). The average specific killing of the JY cells by theCMV-specific CTLs was around 45-50% using an E:T ratio of 10:1 (data notshown). As shown in FIG. 11A, Compound B effectively mediated thekilling of the A431K5 cells (mesothelin-positive HLA-A2-negative), thisspecific killing was 66% in comparison with peptide-loaded JY cells(-150% compared to JY cells). However, when the target A431K5 cells wereincubated with the CMV-specific CTLs alone without preincubation withCompound B or when the target cells were mesothelin-negative (A431cells), with or without preincubation with Compound B, no cytotoxicactivity was observed. Titration experiments which determined thepotency of the fusion protein, shown in FIG. 11B indicate that thekilling of mesothelin-positive A431K5 cells was dose-dependent. Tocompare the cytotoxic activity of Compound B and Compound A fusionproteins, which differ in the length of peptide used to covalentlyattach the antigenic peptide to the β-2 microglobulin, a S35-Methioninerelease assay was performed using similar conditions for both fusionproteins. As shown in FIG. 12, both molecules efficiently andspecifically mediated the killing of A431K5 cells, but not A431 cells.When relatively high concentrations of fusion proteins (Compound B andCompound A) were used, the killing activity of both molecules wassimilar. However, when low concentrations of fusion proteins were usedthe cytotoxic activity of Compound A was superior probably due to betterstability and positioning of the CMV peptide in the MHC peptide-bindinggroove due to the longer linker.

M1-COV/scHLA-A2/SS1

The M1-cov/scHLA-A2/SS1 (scFv) fusion protein was constructed by overlapextension PCR reaction in which the Influenza M1 58-66 peptide and a 15amino acid linker GGGGSGGGGSGGGGS were fused to the N-terminus of thescHLA-A2/SS1(scFv) fusion protein (FIG. 13). The PCR product was ligatedto TA-cloning vector (p-GEM, Promega), transformed to E-coli DH5α cells.Positive colonies were selected and the insert was isolated using EcoRIand Ndel. The insert was ligated to PRB expression vector andtransformed to E-coli DH5α cells. Positive colonies were sent to DNAsequencing (FIG. 14).

Expression and Purification of Compound B

The M1-cov/scHLA-A2/SS1 (scFv) fusion protein was expressed in E. coliBL21 cells and, upon induction with isopropyl β-D-thiogalactoside, largeamounts of recombinant protein accumulated in intracellular inclusionbodies. SDS/PAGE analysis of isolated and purified inclusion bodiesrevealed that the M1-cov/scHLA-A2/SS1 (scFv) fusion protein with thecorrect size constituted 80-90% of the total inclusion bodies mass (FIG.15A). The isolated solubilized inclusion bodies were reduced andrefolded in vitro in a redox-shuffling buffer. Monomeric soluble fusionproteins (M1-cov/scHLA-A2/SS1 (scFv)) were purified by ion-exchangechromatography on Q-sheparose®. SDS/PAGE analysis of theM1-cov/scHLA-A2/SS1 (scFv) fusion proteins revealed a highly purifiedmonomeric molecule with the expected size of 72 KDa (FIG. 15B).

ELISA

To test the binding ability of the purified M1-cov/scHLA-A2/SS1 (scFv)fusion protein to it target antigen, recombinant mesothelin wasimmobilized to immunoplates. The binding of M1-cov/scHLA-A2/SS1 (scFv)fusion protein was monitored by using conformation sensitive mAb W6/32,this anti body recognizes MHC molecules that are folded correctly with apeptide in its groove. As shown in FIG. 16 the binding of theM1-cov/scHLA-A2/SS1 (scFv) fusion protein to recombinant mesothelin wasdose-dependent. This suggests that the two functional domains ofM1-cov/scHLA-A2/SS1 (scFv) fusion protein, the scFv (SS1) domain and theM1-cov/scHLA-A2 domain are folded correctly. Moreover, the scFv (SS1)domain of the fusion protein is in active form and can specifically bindmesothelin.

Flow Cytometry Analysis (FACS)

To test the binding ability of the M1-cov/scHLA-A2/SS1 (scFv) fusionprotein to mesothelin-expressing cell lines, FACS analysis was used. Asa model, target cells that are HLA-A2-negative were used, and theanti-HLA-A2 mAb can be used to monitor the binding of theM1-cov/scHLA-A2/SS1 (scFv) fusion protein to the cell surface antigen.For the FACS analysis, the mesothelin-transfected A431K5 cells wereused. The binding of the M1-cov/scHLA-A2/SS1 (scFv) fusion protein tothe target cells was monitored by anti-HLA-A2 mAb BB7.2 as primaryantibody followed by a FITC-labeled secondary antibody. To test theexpression levels of mesothelin and as positive control, a commercialanti mesothelin mAb K1 was used. As shown in FIG. 17A-17B, A431K5 cellsexpress high levels of mesothelin, whereas the parental A431 cells donot express mesothelin. The cell lines A431 and A431K5 were also testedfor their expression of HLA-A2 using HLA-A2 specific antibody (BB7.2),both cell lines were HLA-A2-negative. However, when A431K5 cells but notA431 cells were pre-incubated with the M1-cov/scHLA-A2/SS1(scFv) fusionproteins, they were positively stained with the HLA-A2-specific antibodyBB7.2 (FIG. 17C-17D). This specific binding of theM1-cov/scHLA-A2/SS1(scFv) fusion protein to A431K5 cells and not to A431cells indicates that the binding is exclusively dependent on theinteraction of the targeting domain (scFv(SS1)) with mesothelin.

Cytotoxicity Assay

To test the ability of the M1-cov/scHLA-A2/SS1 (scFv) fusion protein tomediate the killing of HLA-A2-negative mesothelin-positive cells byHLA-A2-restrictive M158-66 specific CTLs, S35-Methionine release assayusing HLA-A2-negative mesothelin-transfected A431K5 cells was performed.As shown in FIG. 18, M1-cov/scHLA-A2/SS1 (scFv) fusion protein did notmediate the lysis of A431K5 cells (mesothelin-positive HLA-A2-negative).However, the scHLA-A2/SS1(scFv) bearing the M158-66 peptide in itsgroove mediated the killing of mesothelin-positive target cells by theHLA-A2-restricted M158-66 specific CTLs.

Discussion:

This study demonstrates the ability to target covalently linkedpeptide/scMHC/scFv fusion protein to tumor cells can renderHLA-A2-negative cells susceptible to lysis by the relevantHLA-A2-restricted CTLs. As previously shown by Lev et al., and Oved etal. (15,21), this strategy has two major advantages. First, it takesadvantage of the use of recombinant Ab fragments that can localize onthose malignant cells that express a tumor marker, usually associatedwith the transformed phenotype (such as growth factor receptors and/ordifferentiation antigens), with a relatively high degree of specificity.Second, this strategy has the ability to recruit a particular populationof highly reactive cytotoxic T-cells specific to a preselected, highlyantigenic peptide epitope present in the targeted MHC-peptide complex,such as viral-specific T-cell epitopes. This platform approach generatesmultiple molecules with many tumor-specific scFv fragments that targetvarious tumor specific antigens, combined with the ability to targetmany types of MHC-peptide complexes carrying single, preselected, andhighly antigenic peptides derived from tumor, viral, or bacterial T-cellepitopes. These examples present a strategy one step farther by fusingthe 9 amino acid peptide linked by a short linker (20AA) to thepreviously reported scHLA-A2/scFv fusion protein (15AA), and by doingso, stabilizing the peptide in the MHC groove prolonging the generalstability of the fusion proteins. As a model for this new generation offusion proteins, the present invention relates to construction of afusion protein in which the CMV pp65 derived (NLVPMVATV) fused to theN-terminus of the scHLA-A2/SS1 (scFv) molecule and its biochemical andbiological characteristics. It is shown that the two domains of the newfusion protein can refold in vitro to form correctly folded moleculeswith the peptide within the HLA-A2 groove and an active targeting domain(scFv) that can specifically bind its target antigen. Moreover, thisfusion protein had successfully mediated the lysis of HLA-A2-negativemesothelin-positive tumor cells by HLA-A2-restricted CTLs.

Tumor progression is often associated with the secretion ofimmune-suppressive factors and/or the down-regulation of MHC class Iantigen-presentation functions (2). Even when a specific CTL response isdemonstrated in patients, this response is low because the anti-tumorCTL population is rare, very infrequent, and in some cases the CLTs arenot functional or anergic (26). Moreover, it is well-established thatthe number of MHC-peptide complexes on the surface of tumor cells thatpresent a particular tumor-associated peptide is low (27). As shownherein, the new strategy overcame these problems. First, the tumor cellsare coated with MHC-peptide complexes independent of their endogenousMHC expression. Second, the use of tumor specific antigens that areusually part of the tumor phenotype (such as growth factor receptors anddifferentiation antigens) prevent the down regulation of those antigensand prolong the efficiency of the treatment. Third and most important,the effector domain of the fusion protein the MHC-peptide complex canrecruit specific populations of CTLs depending on the peptide harboringthe MHC groove.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the claims. All publications, patents and patent applicationsmentioned in this specification are herein incorporated in theirentirety by reference into the specification, to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

REFERENCES CITED

1. Gilboa, E. How tumors escape immune destruction and what we can doabout it. Cancer Immunol. Immunother. 48, 382-385 (1999).

2. Seliger, B., Maeurer, M. J., & Ferrone, S. Antigen-processingmachinery breakdown and tumor growth. Immunol. Today 21, 455-464 (2000).

3. Garcia-Lora, A., Algarra, I., & Garrido, F. MHC class I antigens,immune surveillance, and tumor immune escape. J. Cell Physiol 195,346-355 (2003).

4. Garrido, F. & Algarra, I. MHC antigens and tumor escape from immunesurveillance. Adv. Cancer Res. 83, 117-158 (2001).

5. McLaughlin, P. et al. Rituximab chimeric anti-CD20 monoclonalantibody therapy for relapsed indolent lymphoma: half of patientsrespond to a four-dose treatment program. J. Clin. Oncol. 16, 2825-2833(1998).

6. Cobleigh, M. A. et al. Multinational study of the efficacy and safetyof humanized anti-HER2 monoclonal antibody in women who haveHER2-overexpressing metastatic breast cancer that has progressed afterchemotherapy for metastatic disease. J. Clin. Oncol. 17, 2639-2648(1999).

7. Pastan, I. & Kreitman, R. J. Immunotoxins in cancer therapy. Curr.Opin. Investig. Drugs 3, 1089-1091 (2002).

8. Boon, T. & van der, B. P. Human tumor antigens recognized byT-lymphocytes. J. Exp. Med. 183, 725-729 (1996).

9. Esche, C., Shurin, M. R., & Lotze, M. T. The use of dendritic cellsfor cancer vaccination. Curr. Opin. Mol. Ther. 1, 72-81 (1999).

10. Offringa, R., van der Burg, S. H., Ossendorp, F., Toes, R. E., &Melief, C. J. Design and evaluation of antigen-specific vaccinationstrategies against cancer. Curr. Opin. Immunol. 12, 576-582 (2000).

11. Wang, E., Phan, G. Q., & Marincola, F. M. T-cell-directed cancervaccines: the melanoma model. Expert. Opin. Biol. Ther. 1, 277-290(2001).

12. Dudley, M. E. et al. Cancer regression and autoimmunity in patientsafter clonal repopulation with antitumor lymphocytes. Science 298,850-854 (2002).

13. Dudley, M. E. & Rosenberg, S. A. Adoptive-cell-transfer therapy forthe treatment of patients with cancer. Nat. Rev. Cancer 3, 666-675(2003).

14. Rosenberg, S. A. Progress in human tumour immunology andimmunotherapy. Nature 411, 380-384 (2001).

15. Lev, A. et al. Tumor-specific Ab-mediated targeting of MHC-peptidecomplexes induces regression of human tumor xenografts in vivo. Proc.Natl. Acad. Sci. U.S.A 101, 9051-9056 (2004).

16. Donda, A. et al. In vivo targeting of an anti-tumor antibody coupledto antigenic MHC class I complexes induces specific growth inhibitionand regression of established syngeneic tumor grafts. Cancer Immun. 3,11 (2003).

17. Ogg, G. S. et al. Sensitization of tumour cells to lysis byvirus-specific CTL using antibody-targeted MHC class I/peptidecomplexes. Br. J. Cancer 82, 1058-1062 (2000).

18. Robert, B., Guillaume, P., Luescher, I., Romero, P., & Mach, J.P.Antibody-conjugated MHC class I tetramers can target tumor cells forspecific lysis by T-lymphocytes. Eur. J. Immunol. 30, 3165-3170 (2000).

19. Robert, B. et al. Redirecting anti-viral CTL against cancer cells bysurface targeting of monomeric MHC class I-viral peptide conjugated toantibody fragments. Cancer Immun. 1, 2 (2001).

20. Savage, P. et al. Anti-viral cytotoxic T-cells inhibit the growth ofcancer cells with antibody targeted HLA class I/peptide complexes inSCID mice. Int. J. Cancer 98, 561-566 (2002).

21. Oved, K., Lev, A., Noy, R., Segal, D., & Reiter, Y.Antibody-mediated targeting of human single-chain class I MHC withcovalently linked peptides induces efficient killing of tumor cells bytumor or viral-specific cytotoxic T-lymphocytes. Cancer Immunol.Immunother. 54, 867-879 (2005).

22. Chang, K. & Pastan, I. Molecular cloning and expression of a cDNAencoding a protein detected by the K1 antibody from an ovarian carcinoma(OVCAR-3) cell line. Int. J. Cancer 57, 90-97 (1994).

23. Chang, K. & Pastan, I. Molecular cloning of mesothelin, adifferentiation antigen present on mesothelium, mesotheliomas, andovarian cancers. Proc. Natl. Acad. Sci. U.S.A 93, 136-140 (1996).

24. Chang, K. & Pastan, I. Molecular cloning of mesothelin, adifferentiation antigen present on mesothelium, mesotheliomas, andovarian cancers. Proc. Natl. Acad. Sci. U.S.A 93, 136-140 (1996).

25. Hassan, R., Bera, T., & Pastan, I. Mesothelin: a new target forimmunotherapy. Clin. Cancer Res. 10, 3937-3942 (2004).

26. Lee, P. P. et al. Characterization of circulating T-cells specificfor tumor-associated antigens in melanoma patients. Nat. Med. 5, 677-685(1999).

27. Christinck, E. R., Luscher, M. A., B arber, B. H., & Williams, D. B.Peptide binding to class I MHC on living cells and quantitation ofcomplexes required for CTL lysis. Nature 352, 67-70 (1991).

28. Jain, R. K. Transport of molecules, particles, and cells in solidtumors. Annu. Rev. Biomed. Eng 1, 241-263 (1999).

29. Bromley, S. K. et al. The immunological synapse. Annu. Rev. Immunol.19, 375-396 (2001).

30. Lanzavecchia, A., Lezzi, G., & Viola, A. From TCR engagement toT-cell activation: a kinetic view of T-cell behavior. Cell 96, 1-4(1999).

31. Chang, K., Pastan, I. & Willingham, M. C. Isolation andcharacterization of a monoclonal antibody, K1, reactive with ovariancancers and normal mesothelium. Int. J. Cancer 50, 373-381 (1992).

32. Chang, K., Pai, L. H., Batra, J. K., Pastan, I. & Willingham, M. C.Characterization of the antigen (CAK1) recognized by monoclonal antibodyK1 present on ovarian cancers and normal mesothelium. Cancer Res. 52,181-186 (1992).

33. Parham, P., Barnstable, C. J. & Bodmer, W. F. Use of a monoclonalantibody (W6/32) in structural studies of HLA-A,B,C, antigens. J.Immunol. 123, 342-349 (1979).

34. Shields, M. J. & Ribaudo, R. K. Mapping of the monoclonal antibodyW6/32: sensitivity to the amino terminus of beta2-microglobulin. TissueAntigens 51, 567-570 (1998).

35. Parham, P. & Brodsky, F. M. Partial purification and some propertiesof BB7.2. A cytotoxic monoclonal antibody with specificity for HLA-A2and a variant of HLA-A28. Hum. Immunol. 3, 277-299 (1981).

What is claimed is:
 1. A fusion protein comprising consecutive aminoacids which, beginning at the amino terminus of the protein, correspondto consecutive amino acids present in (i) a cytomegalovirus humanMHC-restricted peptide, (ii) a first peptide linker, (iii) a human β-2microglobulin, (iv) a second peptide linker, (v) a HLA-A2 chain of ahuman MHC class I molecule, (vi) a third peptide linker, (vii) avariable region from a heavy chain of a scFv fragment of an antibody,and (viii) a variable region from a light chain of such scFv fragment,wherein the consecutive amino acids which correspond to (vii) and (viii)are bound together directly by a peptide bond or by consecutive aminoacids which correspond to a fourth peptide linker and the scFv fragmentis derived from an antibody which specifically binds to mesothelin. 2.The fusion protein of claim 1, wherein the first peptide linker has theamino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6).
 3. The fusionprotein of claim 1, wherein the second peptide linker has the amino acidsequence GGGGSGGGGSGGGGS (SEQ ID NO:8).
 4. The fusion protein of claim1, wherein the third peptide linker has the amino acid sequence ASGG(SEQ ID NO:10).
 5. The fusion protein of claim 1, wherein the fourthpeptide linker has the amino acid sequence GVGGSGGGGSGGGGS (SEQ IDNO:19).
 6. The fusion protein of claim 1, wherein the cytomegalovirushuman MHC-restricted peptide has the amino acid sequence NLVPMVATV (SEQID NO:4).
 7. The fusion protein of claim 1, wherein the sequence of theconsecutive amino acids corresponding to (vii), followed by the fourthpeptide linker, followed by (viii) is set forth in SEQ ID NO:12.
 8. Thefusion protein of claim 1, wherein the consecutive amino acids have theamino acid sequence set forth in SEQ ID NO:2.
 9. A compositioncomprising the fusion protein of claim 1 and a carrier.
 10. Thecomposition of claim 9 wherein the fusion protein is present in thecomposition in a therapeutically effective amount and the carrier is apharmaceutically acceptable carrier.
 11. A nucleic acid constructcomprising a nucleic acid sequence encoding the fusion protein ofclaim
 1. 12. The nucleic acid construct of claim 11, wherein saidnucleic acid sequence is as set forth in SEQ ID NO:1.
 13. A vectorcomprising the nucleic acid construct of claim
 11. 14. An expressionvector comprising the nucleic acid construct of claim 11 and a promoteroperatively linked thereto.
 15. A transformed cell comprising the vectorof claim
 14. 16. An isolated preparation of bacterially-expressedinclusion bodies comprising over 30 percent by weight of the fusionprotein of claim
 1. 17. A process for producing a fusion proteincomprising culturing the transformed cell of claim 15, so that thefusion protein is expressed, and recovering the fusion protein soexpressed.
 18. A method of killing a tumor cell which expressesmesothelin on its surface, the method comprising contacting the tumorcell with the fusion protein of claim 1 in an amount effective toinitiate a CTL-mediated immune response against the tumor cell so as tothereby kill the tumor cell.